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. 2020 Mar 10;10(17):10254-10262.
doi: 10.1039/c9ra10020b. eCollection 2020 Mar 6.

Nobiletin, a citrus polymethoxyflavone, enhances the effects of bicalutamide on prostate cancer cells via down regulation of NF-κB, STAT3, and ERK activation

Yuran Ma  1 , Xiang Ren  1 , Nandini Patel  2 , Xuetao Xu  1   3 , Panpan Wu  1 , Wenfeng Liu  1   3 , Kun Zhang  1   3 , Susan Goodin  4 , Dongli Li  1   3 , Xi Zheng  2   4
Affiliations

Nobiletin, a citrus polymethoxyflavone, enhances the effects of bicalutamide on prostate cancer cells via down regulation of NF-κB, STAT3, and ERK activation

Yuran Ma et al. RSC Adv. .

Abstract

Natural products have shown potential to be combined with current cancer therapies to improve patient outcomes. Nobiletin (NBT) is a citrus polymethoxyflavone and has been shown to exert an anticancer effect in various cancer cells. We investigated the effects and mechanisms of NBT in combination with bicalutamide (BCT), a commonly used anti-androgen drug in prostate cancer therapy, on prostate cancer cells. Our results demonstrate that the combined treatment with NBT and BCT produces an enhanced inhibitory effect on the growth of prostate cancer cells compared to either compound alone. The synergistic action of NBT and BCT was confirmed using isobologram analysis. Moreover, this study has shown that NBT and BCT synergistically inhibited colony formation and migration as well as induced apoptosis. Mechanistic studies demonstrate that NBT and BCT combination reduced key cellular signaling regulators including: p-Erk/Erk, p-STAT3/STAT3 and NF-κB. Overall, these results suggest that NBT combination with BCT may be an effective treatment for prostate cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1. Chemical structures of NBT and BCT.
Fig. 2
Fig. 2. Inhibitory effect of NBT and BCT on the growth of prostate cancer cell lines. Cells were treated with NBT and BCT for 24 h, 48 h, 72 h, and cell viability was measured using MTT assay. Figure showing reduced viability of PC-3 (A) and VCaP (B) upon NBT or BCT exposure. Data shown are mean ± SEM; n = 5.
Fig. 3
Fig. 3. NBT and BCT alone or in combination showing their effect on cell viability in prostate cancer cells. Cell viability was assessed using MTT after treating cells with different concentrations of NBT and/or BCT for 72 h (A). Data shown are mean ± SEM; n = 5. **p < 0.01, ***p < 0.001, significantly different from combination group. Combination index analysis for the cell viability in PC-3 and VCaP cells treated with the combination of NBT and BCT (B). (C) Cell viability in the various cell lines treated with NBT and/or BCT for 72 h by trypan blue exclusion assay. Data shown are mean ± SEM; n = 3. **p < 0.01, ***p < 0.001, significantly different from NBT-treated group, #p < 0.05, ###p < 0.001 significantly different from BCT-treated group. (D) Cell morphology under a microscope treated with NBT and BCT alone or combination. (E) Cell viability of HaCaT cells after treating cells with NBT and/or BCT for 72 h.
Fig. 4
Fig. 4. Effects of NBT and BCT alone or in combination on apoptosis and colony formation of prostate cancer cells. Apoptotic cells were determined by propidium iodide staining (A). Data shown are mean ± SEM; n = 3. ***p < 0.001 between the combination group and the NBT or BCT-treated group. Effect of combined treatment on colony formation. Cell were treated for 24 h and stained with Giemsa on day 5 (B).
Fig. 5
Fig. 5. Effects of NBT and BCT in combination on the migration of prostate cancer cells. For cell migration assays, micrographs were obtained at 0 h and 24 h after scratch. Representative micrographs of cells treated with NBT and/or BCT. Migration of cells was determined and expressed as percentages of the original scratch area. Data shown are mean ± SEM; n = 3. **p < 0.01 between the combination group and the NBT-treated group, #p < 0.05, ##p < 0.05 between the combination group and BCT-treated group.
Fig. 6
Fig. 6. Effect of NBT and BCT in combination on NF-κB activity and the levels of p-p65, Bcl-2 and survivin. For the assay of NF-κB activity, the luciferase activity was measured and expressed as a percentage relative to the control (A). Data shown are mean ± SEM; n = 3. ***p < 0.001 between the combination group and the NBT-treated group, ##p < 0.01, ###p < 0.001 between the combination group and BCT-treated group. Western blotting was performed to determine the levels of p-p65, Bcl-2 and survivin (B).
Fig. 7
Fig. 7. Effect of NBT and BCT on the level of p-Erk1/2, Erk1/2, c-myc, CyclinD1, p-STAT3, STAT3 in PC-3 cell line. Western blotting was used to check the levels of p-Erk1/2, Erk1/2, c-myc, CyclinD1, p-STAT3, STAT3.
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